Highly purified mocarhagin, a cobra venom protease, polynucleotides encoding same and related proteases, and therapeutic uses thereof

ABSTRACT

Methods and compositions for treating a wide range of inflammatory diseases and disorders, and for inhibiting selectin-mediated binding by administering a mocarhagin protein, or fragments thereof with mocarhagin protein activity are provided. The mocarhagin protein may be administered alone, or in combination with a cytokine, lymphokine, or hematopoietic factor.

This application is a continuation-in-part of application Ser. No. 08/012,637, filed Jan. 23, 1998, under the same title in the name of thesame inventors, which was a continuation-in-part of application Ser. No.08/843,373, filed Apr. 15, 1997.

BACKGROUND OF THE INVENTION

Cellular interactions are key to many events in vascular biology. Cellsurface adhesion molecules mediate many of the interactions betweenleukocytes, platelets and the vessel wall. In response to inflammatorystimuli, leukocytes and platelets in the adjacent vasculature initiallyroll on the blood vessel wall, then stick, and finally transmigrate tothe site of insult. The initial rolling event involves a class ofadhesion proteins termed selectins (P-, E-, and L-selectin) whichmediate the interaction between leukocytes, platelets and endothelialcells by their recognition of specific carbohydrate counter-structures,including sialyl-Lewis x. The primary sequence/motif structure of eachof the selectins is similar. Each contains a N-terminal, 118-amino acidcalcium-dependent lectin domain, an EGF motif, a variable number oftandem repetitive motifs related to motifs found in complementregulatory domains, a transmembrane domain and a short cytoplasmic tail.

P-selectin is a 140-kDa integral granule membrane glycoprotein localizedto the α-granules of platelets and the Weibel-Palade bodies ofendothelial cells and is rapidly expressed on both cell types on cellactivation. This suggests that endothelial P-selectin is a criticalmolecule mediating initial adhesion events in acute inflammation, a viewrecently supported by a number of in vivo inflanunatory models includingneutrophil-dependent acute lung injury (Mulligan et al. (1992) J. Clin.Invest. 90, 1600), endotoxin-induced neutropenia (Coughlan et al. (1994)J. Exp. Med. 179, 329), reperfusion injury (Asako et al. (1994) J. Clin.Invest. 93, 1508) and histamine-induced leukocyte rolling in postcapillary venules (Weyrich et al. (1993) J. Clin. Invest. 91, 2620).P-selectin binds to 10,000-20,000 copies of a single class of bindingsites on neutrophils and HL60 cells.

Sako et aL ((1993) Cell 75, 1179) have cloned a ligand for P-selectin,termed P-selectin glycoprotein ligand-1 (PSGL-1) found on the surface ofleukocytes (see also copending application Ser. No. 08/316,305). PSGL-1is a 220 kDa, disulfide-linked homodimeric sialomucin which, whenexpressed by recombinant methodology with the appropriatefucosyltransferase, binds P-selectin, E-slectin and L-selectin in asimilar calcium-dependent manner to the PSGL-1 on neutrophils. PSGL-1has a signal peptide sequence of 17 amino acids followed by a 24-aminoacid PACE cleaved propeptide sequence. The mature N-terminus of PSGL-1contains an unusual stretch of twenty amino acids which is rich innegatively-charged aspartate and glutamate residues and which containsthree tyrosine residues which meet the consensus sequence for0-sulfation by a golgi sulfotransferase. At least one of these tyrosineresidues is sulfated as evaluated by site-directed mutagenesis (Sako etal.).

In addition to binding P-selectin, PSGL-1 also binds L- and E-selectin.In contrast to P-selectin, however, the requirements for E-selectinrecognition are much less rigid. (Spertinit et al., J. Cell. Biol.135:523 (1996)). E-selectin binds a wide variety of sialomucinstructures if they co-express the sialyl-Lewis x structure. L-selectinbinds to a number of different counter-receptors, GLYCAM-1, MadCAM-1 andCD34, which like PSGL-1, are also sialomucins. A major questioncurrently unresolved is what determines selectin specificity in therecognition of specific counter-receptor structures. P-, E- andL-selectin are 60-70% homologous in their N-terminal. 118-amino acidlectin motifs and each similarly recognizes the sialyl-Lewis x andsialyl-Lewis a carbohydrate structures. Further, binding of P-selectinto its receptor on neutrophils is four to five orders of magnitude moreavid than the binding of sialyl-Lewis x. While differences inspecificity and avidity may in part be accounted for by either thepresentation of multiple sialyl-Lewis carbohydrate structures on thereceptor mucin core or by subtle differences in carbohydrate structure,it is probable that the protein component of the sialomucin alsodetermines selectin interaction.

Although the inflammatory response mediated by the P-selectin/PSGL-1interaction is a part of the body's normal defense system, inappropriateinflammatory responses can also result in the development of variousinflammatory disease states. It would, therefore, be desirable toprovide agents for interfering with or blocking the selectin/PSGL-1interaction in order to treat inflammatory disease.

GP1bα is a component of the glycoprotein (GP) Ib-IX complex found on thesurface of platelets and serving as a receptor for von Willibrand factor(vWF). The interaction of the GP Ib-IX complex with vWF mediatesattachment of platelets to the blood vessel wall at the site of injury.It has also can cause aggregation of platelets in high shear conditionsand enable platelet activation at low concentrations of thrombin.

Mocarhagin, a protease found in the venom of cobras (including theMozambiquan spitting cobra, Naja mossambica mossambica, a.k.a. Najamocambique mocambique), has been found to cleave PSGL-1, resulting indisruption of P- and L-selectin mediated cell adhesion. Preparations ofmocarhagin have been reported and demonstrated to serve this purpose.See, U.S. Pat. No. 5,659,018; DeLuca et al., J. Biol. Chem. 270: 26734(1995); Ward et al., Biochem. 35: 4929 (1996). (Spertini et al.)

In addition, it also has been reported that Mocarhagin is capable ofcleaving GP1bα at a position proximal to sulfated tyrosine residueswithin the critical vWF binding domain and disrupting the bindingactivity of GPIbα: DeLuca et al., J. Biol. Chem. 270: 26734 (1995); Donget al., Biochemistry, 33: 13946 (1994).

It is therefore anticipated that an agent that can disrupt thisinteraction may have therapeutic application in a variety of thromboticdisorders such as restenosis and DVT.

However, applicants have discovered that the preparations described inthese documents is only partially purified. Since it is necessary formocarhagin proteins to be provided in highly purified form for suchproteins to be used for therapeutic purposes, it would be desirable toprovide highly purified preparations of mocarhagin proteins.

It would also be desirable to identify and isolate polynucleotidesencoding mocarhagin proteins in order to produce such proteins byrecombinant methods.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising a mocarhaginprotein at least 95% free of other cobra proteins (preferably 95% freeof all other proteins). Preferably, the mocarhagin is homogeneous (i.e.,free of other proteins). In preferred embodiments, the mocarhaginprotein is full-length mocarhagin (as described below). In otherembodiments, the mocarhagin protein is a fragment of full-lengthmocarhagin having mocarhagin proteolytic activity. Preferably, themocarhagin protein is characterized by at least one characteristicselected from the group consisting of:

(a) a molecular weight of approximately 55 kDa under reducingconditions;

(b) a molecular weight of approximately 55 kDa under nonreducingconditions;

(c) an N-terminal amnino acid sequence comprisingTNTPEQDRYLQAKKYIEFYVVVDNVMYRKY; (SEQ ID NO:1)

(d) mocarhagin proteolytic activity;

(e) the ability to inhibit platelet binding to vWF;

(f) requirement of calcium ion for activity;

(g) requirement of zinc ion for activity;

(h) an activity substantially inhibited by excess EDTA; and

(i) an activity substantially inhibited by high concentrations of DFP.

In some embodiments, the mocarhagin protein has the N-terminal sequences(SEQ ID NO:2) TNTPEQDRYLQAKKYIEFYVVVDNVMYRKYTGKLHVITXXVYEMNALN.

In particularly preferred embodiments, the mocarhagin protein is capableof cleaving capable of cleaving a material selected from the groupconsisting of anionic polypeptides containing sulfated tyrosineresidues, PSGL-1 and GP1bα. PSGL-1 and/or GP1bα. Compositions comprisinga therapeutically effective amount of a mocarhagin protein and apharmaceutically acceptable carrier are also provided.

Methods of treating an inflammatory disease and thrombotic disorders andof inhibiting selectin-mediated binding comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising a mocarhagin protein to a mammalian subject are disclosed.

The invention also provides a method of isolating mocarhagin from venom,said method comprising:

(a) subjecting a composition comprising cobra venom to a heparinaffinity chromatography column;

(b) subjecting the eluate from said heparin affinity column to a sizeexclusion column. (c) subjecting the eluate from said size exclusioncolumn to a Mono S column; and

(d) eluting said mocarhagin from said Mono S column. Compositionscomprising a protein isolated according to these methods (and optionallyfurther comprising a pharmaceutically acceptable carrier) are alsoencompassed by the claimed invention. Such compositions can also be usedin methods of treating an inflammatory disease and of inhibitingselectin-mediated binding which comprise administering a therapeuticallyeffective amount of such compositions to a mammalian subject.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:6;    -   (b) the amino acid sequence of SEQ ID NO:6 from amino acid 24 to        amino acid 621;    -   (c) the amino acid sequence of SEQ ID NO:6 from amino acid 192        to amino acid 621;    -   (d) fragments of the amino acid sequence of SEQ ID NO:6 encoding        a protein having mocarhagin activity; and    -   (e) the amino acid sequence encoded by the cDNA insert of clone        NMM-1 deposited under accession number ATCC 209588; the protein        being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:5;    -   (b). a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:5 from nucleotide 78 to nucleotide 1940;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:5 from nucleotide 147 to nucleotide 1940;    -   (d) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:5 from nucleotide 651 to nucleotide 1940;    -   (e) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-1 deposited under accession number ATCC        209588;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:6;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:6 from amino acid 24 to amino acid        621;    -   (h) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:6 from amino acid 192 to amino acid        621;    -   (i) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:6 encoding a protein having        mocarhagin activity;    -   (j) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(h) above;    -   (k) a polynucleotide which encodes a species homologue of the        protein of (f), (g) or (h) above: and    -   (l) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:8;    -   (b) the amino acid sequence of SEQ ID NO:8 from amino acid 24 to        amino acid 439;    -   (c) the amino acid sequence of SEQ ID NO:8 from amino acid 192        to amino acid 439;    -   (d) fragments of the amino acid sequence of SEQ ID NO:8 encoding        a protein having mocarhagin activity; and    -   (e) the amino acid sequence encoded by the cDNA insert of clone        NMM-2 deposited under accession number ATCC 209589;        the protein being substantially free from other mammalian        proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:7;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:7 from nucleotide 85 to nucleotide 1401;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:7 from nucleotide 154 to nucleotide 1401;    -   (d) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:7 from nucleotide 658 to nucleotide 1401;    -   (e) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-2 deposited under accession number ATCC        209589;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:8;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:8 from amino acid 24 to amino acid        439;    -   (h) a polynucleolide encoding a protein comprising the amino        acid sequence of SEQ ID NO:8 from amino acid 192 to amino acid        439;    -   (i) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:8 encoding a protein having        mocarhagin activity;    -   (j) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(h) above;    -   (k) a polynucleotide which encodes a species homologue of the        protein of (f), (g) or (h) above; and    -   (1) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:10;    -   (b) the amino acid sequence of SEQ ID NO:10 from amino acid 24        to amino acid 613;    -   (c) the amino acid sequence of SEQ ID NO:10 from amino acid 192        to amino acid 613;    -   (d) fragments of the amino acid sequence of SEQ ID NO:10        encoding a protein having mocarhagin activity; and    -   (e) the amino acid sequence encoded by the cDNA insert of clone        NMM-9 deposited under accession number ATCC 209586;        the protein being substantially free from other mammalian        proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9 from nucleotide 67 to nucleotide 1905;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9 from nucleotide 136 to nucleotide 1905;    -   (d) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9 from nucleotide 640 to nucleotide 1905;    -   (e) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-9 deposited under accession number ATCC        209586;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:10;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:10 from amino acid 24 to amino acid        613;    -   (h) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:10 from amino acid 192 to amino acid        613;    -   (i) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:10 encoding a protein        having mocarhagin activity;    -   (j) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(h) above;    -   (k) a polynucleotide which encodes a species homologue of the        protein of (f), (g) or (h) above; and    -   (l) a polynucleotide which hybridizes under stringent conditions        to a polynucieotide of (a)-(h) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:12;    -   (b) the amino acid sequence of SEQ ID NO:12 from amino acid 24        to amino acid 521;    -   (c) the amino acid sequence of SEQ ID NO:12 from amino acid 192        to amino acid 521;    -   (d) fragments of the amino acid sequence of SEQ ID NO:12        encoding a protein having mocarhagin activity; and    -   (e) the amino acid sequence encoded by the cDNA insert of clone        NMM-12 deposited under accession number ATCC 209585;        the protein being substantially free from other mammalian        proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:11;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:11 from nucleotide 78 to nucleotide 1640;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:11 from nucleotide 147 to nucleotide 1640;    -   (d) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:11 from nucleotide 651 to nucleotide 1640;    -   (e) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NIM-12 deposited under accession number        ATCC 209585;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:12;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:12 from amino acid 24 to amino acid        521;    -   (h) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:12 from amino acid 192 to amino acid        521;    -   (i) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:12 encoding a protein        having mocarhagin activity;    -   (j) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(h) above;    -   (k) a polynucleotide which encodes a species homologue of the        protein of (f), (g) or (h) above; and    -   (I) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:14;    -   (b) the amino acid sequence of SEQ ID NO:14 from amino acid 24        to amino acid 592;    -   (c) the amino acid sequence of SEQ ID NO:14 from amino acid 192        to amino acid 592;    -   (d) fragments of the amino acid sequence of SEQ ID NO:12        encoding a protein having mocarhagin activity; and    -   (e) the amino acid sequence encoded by the cDNA insert of clone        NMM-13 deposited under accession number ATCC 209584; the protein        being substantially free from other mammalian proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:13;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:13 from nucleotide 83 to nucleotide 1858;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:13 from nucleotide 152 to nucleotide 1858;    -   (d) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:13 from nucleotide 656 to nucleotide 1858;    -   (e) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-13 deposited under accession number        ATCC 209584;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:14;    -   (g) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:14 from amino acid 24 to amino acid        592;    -   (h) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:14 from amino acid 192 to amino acid        592;    -   (i) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:14 encoding a protein        having mocarhagin activity;    -   (j) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(h) above;    -   (k) a polynucleotide which encodes a species homologue of the        protein of (f), (g) or (h) above; and    -   (l) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(h) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:16;    -   (b) the amino acid sequence of SEQ ID NO:16 from amino acid 62        to amino acid 462;    -   (c) fragments of the amino acid sequence of SEQ ID NO:16        encoding a protein having mocarhagin activity; and    -   (d) the amino acid sequence encoded by the cDNA insert of clone        NMM-3 deposited under accession number ATCC 209587;        the protein being substantially free from other mammalian        proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:15;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:15 from nucleotide 3 to nucleotide 1388;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:15 from nucleotide 186 to nucleotide 1388;    -   (d) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-3 deposited under accession number ATCC        209587;    -   (e) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:16;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:16 from amino acid 62 to amino acid        462;    -   (g) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:16 encoding a protein        having mocarhagin activity;    -   (h) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(g) above;    -   (i) a polynucleotide which encodes a species homologue of the        protein of (e) or (f) above; and    -   (j) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(g) above.

The present invention also provides a composition comprising amocarhagin protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:18;    -   (b) the amino acid sequence of SEQ ID NO:18 from amino acid 197        to amino acid 621;    -   (c) fragments of the amino acid sequence of SEQ ID NO:18        encoding a protein having mocarhagin activity; and    -   (d) the amino acid sequence encoded by the cDNA insert of clone        NMM-9ek deposited under accession number ATCC 209583;        the protein being substantially free from other mammalian        proteins.

Yet other embodiments provide for a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:17;    -   (b) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:17 from nucleotide 67 to nucleotide 1929;    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:17 from nucleotide 655 to nucleotide 1929;    -   (d) a polynucleotide encoding the mature protein encoded by the        cDNA insert of clone NMM-9ek deposited under accession number        ATCC 209583;    -   (e) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:18;    -   (f) a polynucleotide encoding a protein comprising the amino        acid sequence of SEQ ID NO:18 from amino acid 197 to amino acid        621;    -   (g) a polynucleotide encoding a protein comprising a fragment of        the amino acid sequence of SEQ ID NO:18 encoding a protein        having mocarhagin activity;    -   (h) a polynucleotide which is an allelic variant of a        polynucleotide of (a)-(g) above;    -   (i) a polynucleotide which encodes a species homologue of the        protein of (e) or (f) above; and    -   (j) a polynucleotide which hybridizes under stringent conditions        to a polynucleotide of (a)-(g) above.

Compositions comprising an antibody which specifically reacts with themocarhagin proteins or a fragments thereof having mocarhagin proteolyticactivity are also provided.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts an SDS-PAGE gel analysis of fractions containingmocarhagin eluted from the size exclusion column as described herein.Multiple protein species of similar molecular weight can be seen inthese fractions.

FIG. 2 depicts an SDS-PAGE gel analysis of fractions containingmocarhagin eluted from the Mono-S column as described herein. This geldemonstrates the high degree of purity of the mocarhagin materialpurified as described in Example 1.

FIG. 3 is an SDS-PAGE gel analysis of fractions containingenterokinase-cleaved mocarhagin protein produced by expression of theNMM-9ek construct described below. The dot indicates the novel ˜50 kband produced by enterokinase cleavage.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention provides a highly specific metalloproteinase,mocarhagin, which has been purified from the venom of the Mozambiquanspitting cobra, Naja mossambica mossambica (a.k.a., Naja mocambiquemocambique). Mocarhagin cleaves a ten amino acid peptide from the matureN-terminus of PSGL-1 and abolishes the ability of PSGL-1 to bindP-selectin. These results are in accord with the negativecharge/sulfated tyrosine cluster at the N-terminus of PSGL-1 being animportant determinant of P-selectin recognition in addition to therecognition of carbohydrate structure.

Mocarhagin can be purified from cobra venom according to the methoddescribed in the examples below. Other methods of purifying mocarhaginfrom cobra venom will also be apparent to those skilled in the art. Theprogress of any purification scheme for mocarhagin can be monitored onthe basis of the biochemical characteristics of mocarhagin describedherein and the assays for PSGL-1 digestion and neutrophil/HL60 cellbinding described below.

A cDNA encoding a mocarhagin protein (“clone NMM-1”) has also beencloned from a cobra venom gland library as described in Example 5 below.The nucleotide sequence of the NMM-1 cDNA is reported as SEQ ID NO:5.Clone NMM-1 was deposited with the American Type Culture Collection onJan. 16, 1998 at accession number ATCC 209588. The protein sequenceencoded by clone NMM-1 is reported as SEQ ID NO:6. Amino acids 1-23 ofSEQ ID NO:6 are a predicted signal peptide. The mocarhagin propeptidebegins with amino acid 24, with the mature protein beginning at aminoacid 192.

Four additional full-length cDNAs encoding closely related proteases(clones “NMM-2”, “NMM-9”, “NMM-12” and “NMM-13”) were also isolated fromthe cobra venom gland library as described in Example 5 below. Each ofthe proteins encoded by such cDNAs is also a “mocarhagin protein” asused herein.

The nucleotide sequence of the clone NMM-2 cDNA is reported as SEQ IDNO:7. Clone NMM-2 was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209589. The proteinsequence encoded by clone NMM-2 is reported as SEQ ID NO:8. Amino acids1-23 of SEQ ID NO:8 are a predicted signal peptide. The mocarhaginpropeptide begins with amino acid 24, with the mature protein beginningat amino acid 192.

The nucle6tide sequence of the clone NMM-9 cDNA is reported as SEQ IDNO:9. Clone NMM-9 was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209586. The proteinsequence encoded by clone NMM-2 is reported as SEQ ID NO:10. Amino acids1-23 of SEQ ID NO:10 are a predicted signal peptide. The mocarhaginpropeptide begins with amino acid 24, with the mature protein beginningat amino acid 192.

The nucleotide sequence of the clone NMM-12 cDNA is reported as SEQ IDNO:11. Clone NMM-12 was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209585. The proteinsequence encoded by clone NMM-12 is reported as SEQ ID NO:12. Aminoacids 1-23 of SEQ ID NO:12 are a predicted signal peptide. Themocarhagin propeptide begins with amino acid 24, with the mature proteinbeginning at amino acid 192.

The nucleotide sequence of the clone NMM-13 cDNA is reported as SEQ IDNO:13. Clone NMM-13 was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209584. The proteinsequence encoded by clone NMM-13 is reported as SEQ ID NO:14. Aminoacids 1-23 of SEQ ID NO:14 are a predicted signal peptide. Themocarhagin propeptide begins with amino acid 24, with the mature proteinbeginning at amino acid 192.

Two additional partial cDNAs encoding other closely related proteases(clones “NMM-3” and “NMM-10”) were also isolated from the cobra venomgland library as described in Example 5 below. Each of the proteinsecnoded by such cDNAs is also a “mocarhagin protein” as used herein.

The nucleotide sequence of the clone NMM-3 cDNA is reported as SEQ IDNO:15. Clone NMM-3 was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209587. The proteinsequence encoded by clone NMM-3 is reported as SEQ ID NO:16. Amino acids1-61 of SEQ ID NO:16 are part of the propeptide sequence. The maturemocarhagin propeptide begins with amino acid 62.

Applicants have also discovered that removal of the mocarhaginpropeptide increases the catalytic activity of the enzyme. Thusengineered recombinant forms of mocarhagin include forms havingendopeptidase cleavage sites between the propeptide segment and maturepeptide segment, including but not limited to, enterokinase cleavagesites or PACE cleavage sites. Alternatively, a propeptide or secretorysignal peptide may be substituted for the native mocarhagin propeptideto enable the secretion of active recombinant mocarhagin from eucaryotichost cells.

The NMM-9 cDNA was used to make a modified construct which includes anenterokinase cleavage sight. Certain preferred embodiments of thepresent invention included such an enterokinase cleavage site in orderto increase production of active (i.e., properly cleaved to remove theleader sequence) protein. The nucleotide sequence of the constructcontaining the celavage site, clone NMM-9ek, is reported as SEQ IDNO:17. Clone NMM-9ek was deposited with the Americal Type CultureCollection on Jan. 16, 1998 at accession number ATCC 209583. The proteinsequence encoded by clone NMM-9ek is reported as SEQ ID NO:18. Theenterokinase cleavage site is found at amino acid 192-196 of SEQ IDNO:18. Amino acids 1-196 of SEQ ID NO:18 are part of the propeptidesequence which is cleaved upon enterokinase treatment. The maturecleaved mocarhagin propeptide begins with amino acid 197.

For the purposes of the present invention, a protein is defined ashaving “mocarhagin proteolytic activity” when (1) it digests PSGL-1,such as in the PSGL-1 digestion assay described in Example 3 below,and/or (2) inhibits the binding of P-selectin to neutrophils or HL60cells, such as in the binding inhibition assay described in Example 2below, and/or (3) cleaves a peptide derived from PSGL-1(pyroEATEYEYLDYDFLPE, SEQ ID NO:3), such as in the peptide cleavageassay described in Example 4 below. Preferably, in the PSGL-1 digestionassay complete cleavage of ³⁵[S]-sPSGL-1.T7 is achieved in 20 min. using10 μg/mL mocarhagin protein; more preferably in 20 min. using less than1 μg/mL mocarhagin protein. Preferably, in the neutrophil/HL 60 bindinginhibition assay the mocarhagin protein exhibits an IC₅₀ of less thanabout 100 μg/mL, more preferably less than about 1 μg/mL.

Fragments of mocarhagin having mocarhagin proteolytic activity are alsoencompassed by the present invention. Fragments of mocarhagin havingmocarhagin proteolytic activity can be identified by the PSGL-1digestion assay and neutrophil/HL60 binding inhibition assay describedbelow. Fragments of mocarhagin may be in linear form or they may becyclized using known methods, for example, as described in H.U.Saragovi, et aL, Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et aL, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. For the purposes of thepresent invention, all references to “mocarhagin protein” herein includemocarhagin and fragments having mocarhagin proteolytic activity.

Isolated mocarhagin protein may be useful in treating conditionscharacterized by P- or E-selectin mediated intercellular adhesion oradhesion events mediated by GP1bα, including without limitation thoseinvolving platelet aggregation. Such conditions include, withoutlimitation, myocardial infarction, vessel restenosis, thrombosis,bacterial or viral infection, metastatic conditions, inflammatorydisorders such as arthritis, acute respiratory distress syndrome,asthma, emphysema, delayed type hypersensitivity reaction, systemiclupus erythematosus, thermal injury such as bums or frostbite,autoimmune thyroiditis, experimental allergic encephalomyelitis,multiple sclerosis, multiple organ injury syndrome secondary to trauma,diabetes, Reynaud's syndrome, neutrophilic dermatosis (Sweet'ssyndrome), inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's disease, necrotizingenterocolitis, granulocyte transfusion associated syndrome,cytokine-induced toxicity, and the like. Mocarhagin protein may also beuseful in organ transplantation, both to prepare organs fortransplantation and to quell organ transplant rejection. Mocarhaginprotein may be used to treat hemodialysis and leukophoresis patients.Mocarhagin protein may be used itself as an inhibitor of P- orE-selectin-mediated intercellular adhesion or to design inhibitors of P-or E-selectin-mediated intercellular adhesion. The present inventionencompasses both pharmaceutical compositions containing mocarhaginprotein and therapeutic methods of treatment or use which employmocarhagin protein.

Mocarhagin protein may be used as a pharmaceutical composition whencombined with a pharmaceutically acceptable carrier. Such a compositionmay contain, in addition to mocarhagin protein and carrier, diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswell known in the art. The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1,IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, L-11, IL-12,G-CSF, Meg-CSF, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may contain thrombolytic or anti-thrombotic factors such asplasminogen activator and Factor VM. The pharmaceutical composition mayfurther contain other anti-inflammatory agents. Such additional factorsand/or agents may be included in the pharmaceutical composition toproduce a synergistic effect with mocarhagin protein, or to minimizeside effects caused by the mocarhagin protein. Conversely, mocarhaginprotein may be included in formulations of the particular cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent to minimize side effects of thecytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which mocarhagin protein is combined, in addition to otherpharmaceutically acceptable carriers, with amphipathic agents such aslipids which exist in aggregated form as micelles, insoluble monolayers,liquid crystals, or lamellar layers which in aqueous solution. Suitablelipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Preparation of such liposomalformulations is within the level of skill in the art, as disclosed, forexample, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat.No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which areincorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,healing of chronic conditions characterized by P-selectin- orE-selectin-mediated or GP1bα-mediated cellular adhesion or increase inrate of healing of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of mocarhagin protein is administeredto a mammal having a P-selectin-mediated or GP1bα-mediated diseasestate. Mocarhagin protein may be administered in accordance with themethod of the invention either alone or in combination with othertherapies such as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, isolated mocarhagin proteinmay be administered either simultaneously with the cytokine(s),lymphokine(s), other hematopoietic factor(s). thrombolytic oranti-thrombotic factors, or sequentially. If administered sequentially,the attending physician will decide on the appropriate sequence ofadministering isolated mocarhagin protein in combination withcytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors.

Administration of mocarhagin protein used in the pharmaceuticalcomposition or to practice the method of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, or cutaneous, subcutaneous, or intravenous injection.Intravenous administration to the patient is preferred.

When a therapeutically effective amount of mocarhagin protein isadministered orally, mocarhagin protein will be in the form of a tablet,capsule, powder, solution or elixir. When administered in tablet form,the pharmaceutical composition of the invention may additionally containa solid carrier such as a gelatin or an adjuvant. The tablet, capsule,and powder contain from about 5 to 95% mocarhagin protein, andpreferably from about 25 to 90% mocarhagin protein. When administered inliquid form, a liquid carrier such as water, petroleum, oils of animalor plant origin such as peanut oil, mineral oil, soybean oil, or sesameoil, or synthetic oils may be added. The liquid form of thepharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of mocarhagin protein and preferablyfrom about 1 to 50% mocarhagin protein.

When a therapeutically effective amount of mocarhagin protein isadministered by intravenous, cutaneous or subcutaneous injection,mocarhagin protein will be in the form of a pyrogen-free, parenterallyacceptable aqueous solution. The preparation of such parenterallyacceptable protein solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection should contain, in addition to mocarhagin protein an isotonicvehicle such as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection, or other vehicle as known in the art. The pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additive known to thoseof skill in the art.

The amount of mocarhagin protein in the pharmaceutical composition ofthe present invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments which thepatient has undergone. Ultimately, the attending physician will decidethe amount of mocarhagin protein with which to treat each individualpatient. Initially, the attending physician will administer low doses ofmocarhagin protein and observe the patient's response. Larger doses ofmocarhagin protein may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.1 μg to about 100 mg of mocarhagin protein per kg bodyweight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the mocarhagin protein will be in therange of 12 to 24 hours of continuous intravenous administration.Ultimately the attending physician will decide on the appropriateduration of intravenous therapy using the pharmaceutical composition ofthe present invention.

Mocarhagin protein of the invention may also be used to immunize animalsto obtain polyclonal and monoclonal antibodies which specifically reactwith the mocarhagin protein and which may inhibit P-selectin-mediated orGP1bα-mediated cellular adhesion. Such antibodies may be obtained usingthe entire mocarhagin protein as an immunogen, or by using fragments ofmocarhagin protein such as the soluble mature mocarhagin protein.Smaller fragments of the mocarhagin protein may also be used to immunizeanimals. The peptide immunogens additionally may contain a cysteineresidue at the carboxyl terminus, and are conjugated to a hapten such askeyhole limpet hemocyanin (KLH). Additional peptide immunogens may begenerated by replacing tyrosine residues with sulfated tyrosineresidues. Methods for synthesizing such peptides are known in the art,for example, as in R. P. Merrifield, J.Amer.Chem.Soc. 85, 2149-2154(1963); J. L. Krstenansky, etal, FEBS Lett. 211, 10 (1987).

Also included in the invention are isolated DNAs which hybridize to theDNA sequence set forth in SEQ ID NO:5 under stringent (e.g. 4×SSC at 65°C. or 50% formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or30-40% formamide at 42° C.) conditions.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the mocarhagin proteins recombinantly. Manysuitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein “operably linked” means enzymatically or chemicallyligated to form a covalent bond between the isolated polynucleotide ofthe invention and the expression control sequence, in such a way thatthe mocarhagin protein is expressed by a host cell which has beentransformed (transfected) with the ligated polynucleotide/expressioncontrol sequence.

A number of types of cells may act as suitable host cells for expressionof the mocarhagin protein. Suitable host cells are capable of attachingcarbohydrate side chains characteristic of functional mocarhaginprotein. Such capability may arise by virtue of the presence of asuitable glycosylating enzyme within the host cell, whether naturallyoccurring, induced by chemical mutagenesis, or through transfection ofthe host cell with a suitable expression plasmid containing apolynucleotide encoding the glycosylating enzyme. Host cells include,for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, humankidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3cells, CV-1 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, or HaKcells.

The mocarhagin protein may also be produced by operably linking theisolated polynucleotide of the invention to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

Alternatively, it may be possible to produce the mocarhagin protein inlower eukaryotes such as yeast, fungi or in prokaryotes such as bacteriaPotentially suitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. Suitable fungi strains include Aspergillus sp.orany fungi strain capable of expressing heterologous proteins.

The mocarhagin protein of the invention may also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide encoding themocarhagin protein.

The mocarhagin protein of the invention may be prepared by culturingtransformed host cells under culture conditions necessary to express amocarhagin protein of the present invention. The resulting expressedprotein may then be purified from culture medium or cell extracts asdescribed in the examples below.

Alternatively, the mocarhagin protein of the invention is concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred(e.g., S-Sepharose® columns). The purification of the mocarhagin proteinfrom culture supernatant may also include one or more column steps oversuch affinity resins as concanavalin A-agarose, heparin-toyopearl® orCibacrom blue 3GA Sepharose®; or by hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or by immunoaffinity chromatography.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the mocarhagin protein. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein. The mocarhagin protein thus purified is substantially free ofother mammalian or other host cell proteins and is defined in accordancewith the present invention as “isolated mocarhagin protein”.

EXAMPLE

The following examples are presented to illustrate, not to limit, thepresent invention.

Example 1 Purification of Mocarhagin

20 grams of crude protein from snake venom (Naja mossambica mossambica,Sigma, product no. V1627) were dissolved in 500 mL deionized H₂O andcentrifuged at 10 K rpm for thirty minutes at 4 C. The supernatant wasloaded onto a 200 mL Heparin−650 M affinity column (Toyopearl, Tosohaas)equilibrated with 50 mm Tris-HCl ph 7.6 (buffer A) and 0.2M NaCl. Thecolumn was first washed extensively (to baseline) and mocarhagin waseluted with a gradient of 0.2-1.0 M NaCl in buffer A. Fractionscontaining the protease as monitored by SDS-PAGE (band with molecularweight ˜55 kD) were pooled, concentrated using bentriprep-10 (Amicon)and applied to 21.5 mm ID×60 CM size exclusion column (G 3000SW,Tosohaas) in PBS at RT. Fractions eluted from the size exclusion columnwere analyzed by SDS-PAGE (FIG. 1), which showed the presence ofmultiple proteins of similar molecular weight.

Fractions containing mocarhagin were pooled and applied onto a Mono S10110 column (Pharmacia) equilibrated in 50 mm HEPES ph 8.0 (buffer B)at RT a O-IM NaCl in buffer B, gradient was used to elute the protein.The fractions were assayed by SDS-PAGE, pooled and frozen at 80 C. Therecovery was 2-3 mg of mocarahagin per gram snake venom processed with apurity greater than 95%. FIG. 2 depicts a gel demonstrating the purityof the mocarhagin produced as herein described.

The N-terminal sequence was determined for the process described aboveas TNTPEQDRYLQAKKYIEFYVVVDNVMYRKYTGKLHVITXXVYEMNALN (SEQ ID NO:2). Theresidues indicated in caps (TNTPEQDRYLQAKKYIEFYVVVDNVMYRKY, SEQ ID NO:1)were determined to a higher degree of certainty.

Example 2 Neutrophil/HL60 Binding Inhibition Assay

Neutrophils were isolated from venous blood anticoagulated with heparin(20 units/mL, final concentration) according to the method of Bignoldand Ferrante ((1987) J. Immunol. Meth. 96, 29). The neutrophilswere >95% pure as evaluated by flow cytometry and >98% viable by trypanblue exclusion. HL60 cells were cultured in RPMI medium supplementedwith 10% fetal calf serum. Immediately before use, cells were washedtwice with phosphate-buffered saline (0.01 M sodium phosphate, 0.15 Msodium chloride, pH 7.4). Neutrophils and cultured cells were finallyresuspended at 2×10⁷/mL in RPMI medium supplemented with 1% fetal calfserum. Binding of ¹²⁵I-labeled P-selectin (Skinner et al.) toneutrophils or HL60 cells was evaluated by incubating ¹²⁵I-labeledP-selectin (0.5 μg/mL, final concentration) with cells (1×10⁷/mL, finalconcentration) at 22° C. in a final volume of 200 μl. After 30 min,duplicate 50 μl aliquots were withdrawn and loaded onto 200 μl of 17%(w/v) sucrose in RPMI medium containing 1% fetal calf serum. Neutrophilswere pelleted at 8,750×g for 2 min. After careful aspiration of thesupernatant, radiolabel associated with the cell pellets was measured ina -counter. Nonspecific binding of ¹²⁵I-labeled P-selectin was assessedusing a 50-fold excess of unlabeled P-selectin (Skinner et al.).

To examine the effect of pretreatment of neutrophils or HL60 cells withmocarhagin on P-selectin binding, washed cells (2×10⁷./mL) in RPMI made1% in fetal calf serum were incubated in the presence or absence of 10mM EDTA followed by mocarhagin (0.025-100 μg/mL, final concentrations)for 30 min at 22C. P-selectin binding was then either directly assessedor was assessed after centrifugation of the cells, which were thenwashed twice and finally resuspended in RPMI with 1% fetal calf serum.In some experiments, DFP-treated mocarhagin was employed in place ofmocarhagin. To evaluate the effect of supernatant from mocarhagintreated cells on P-selectin binding, HL60 cells at 10⁸/mL in 0.01 MTris, 0.015 M sodium chloride, 0.001 M calcium chloride, pH 7.4, wereincubated with mocarhagin (12 μg/mL) for 10 min at 22° C. Thesupernatant collected following centrifugation at 1000×g for 10 min wasmade 0.1% in BSA and loaded onto a heparin Sepharose CL-6B column (0.5×5cm) to remove mocarhagin. The flow through was then tested for itseffect on P-selectin binding to HL60 cells.

Example 3 PSGL-1 Digestion Assay

COS cells were cotransfected with three plasmids encoding soluble PSGL-1(pED.sPSGL-1.T7; Sako et al.), alpha 1,3/1,4 fucosyltransferase(pEA.3/4FT) and soluble PACE (pEA-PACE SOL; Wasley et al. (1993) J.Biol. Chem. 268, 8458-8465). [³⁵S]Methionine-labeled COS conditionedmedium containing sPSGL-1.T7 was digested with 5 μg/mL mocarhagin inTBS, 2 mM calcium chloride; 1 mg/mL BSA for 20 min at 37C. The abilityof sPSGL-1.T7 to bind P-selectin was assessed by precipitation with theP-selectin IgG chimera LECγI (Sako et al.) preabsorbed onto protein ASepharose beads in TBS, 2 mM calcium chloride, 1 mg/mL BSA for 4 h at4C. A control experiment was also performed where the LECγI protein ASepharose beads were pre-treated with mocarhagin and then exhaustivelywashed prior to presentation of sPSGL-1.T7. For immunoprecipitationanalysis of untreated and mocarhagin treated sPSGL-1.T7, the proteasewas inactivated by the addition of 5 mM EDTA. sPSGL-1.T7 was thenimmunoprecipitated with anti-PSGL-1 polyclonal antibodies Rb3026 (raisedagainst COS produced sPSGL-1.T7; Sako et al.) or Rb3443 (raised againstthe N-terminal peptide of PACE cleaved PSGL-1:QATEYEYLDYDFLPE, SEQ IDNO:4).

Example 4 Peptide Cleavage Assay

A digestion buffer (10 mM MOPS, 150 mM NaCl, 1 mM CaCl₂; 1 mM MgCl₂,0.02% NaN₃, pH 7.5) and a peptide substrate solution(pyroEATEYEYLDYDFLPE (SEQ ID NO:3), 10 mM in DMSO) were prepared. 2.5 μLpeptide substrate solution (250 pM final substrate concentration) wascombined with mocarhagin sample material (10 μg/mL final mocarhaginconcentration) and adjusted to 100 μL with digestion buffer using noless than 75 pL. This mixture was digested at 37C for 16 hours inparallel with a control sample (no mocarhagin added).

50 μL aliquots of the digested samples were run on an RP-HPLC column(Vydac C18 218TP54, 4.6×250 mm), using the following solvents: solventA, 0.1% TFA in H₂O; solvent B, 0.075% TFA in 90% AcN; flow rate 1mL/min. The presence of peptides in the eluate was measured byabsorbance at 214 nm, 260 nm and 280 nm. A positive assay result wasindicated by observing elution of two peptide peaks in the tested sanplewhich both elute earlier than the single peptide peak observed in thenegative control.

Example 5 Cloning of Polynucleotide Encoding Mocarhaoin Protein

Venom glands from five Mozambiquan spitting cobras, Naja mossambicamossambica, were dissected at two hour intervals, two to twelve hoursfollowing stimulation of venom production. Poly A+RNA was isolated fromtotal RNA of the pooled gland tissue using an Oligotex Direct MRNA kit(Qiagen, Chatsworth, Calif.). Synthesis of cDNA was performed usingSuperscript Choice System (Gibco BRL, Gaithersberg, Md.) using oligo dTand random hexamer primers, EcoRI adapters. The cDNA was ligated withEcoRI digested lambda Zap II cloning vector (Stratagene, La Jolla,Calif.).

Using the above cDNA preparation as template, a PCR reaction wasperformed using degenerate oligonucleotides based on the N-terminal 30residue amino acid sequence described above. The sequences of theforward primer consisted of 5′-ACNCCNGARCARGAY (SEQ ID NO:19). Thesequences of the reverse primer consisted of 5′-RTAYTTYCKRTACAT (SEQ IDNO:20). A resulting 84 bp product was subsequently identified and DNAsequencing confirmed the sequence encoded 30 amino acid residues havinga high degree of homology to the previously determined amino acidsequence. Two oligonucleotides 24 nucleotides in length,5′-CAGGACAGGTACTTGCAGGCCAAA (SEQ ID NO:21) and 5′-ATCGAGTTTTACGTGGTTGTGGAC (SEQ ID NO:22), were synthesized based on thePCR product sequence and used as ³²p hybridization probes to screenapproximately 10⁶ plaques of plated lambda Zap II library. Duplicatesets of Duralose filters (Stratagene, La Jolla, Calif.) were hybridizedseperately with each ³²p hybridization probe in 5×SSC, 5×Denhardt's,0.1% SDS, 50ug/rnl yeast tRNA 16hrs @40C. Filters were washed with4×SSC, 0.1% SDS @ room temperature. then twice at 45C for 30min.Autoradiography was −70C overnight with intensifying screen. Plaquesshowing positive hybridization to both probes were isolated andultimately characterized by nucleotide sequencing.

Clones NMM-1, NMM-2, NMM-3, NMM-9, NMM-10, NMM-12 and NMM-13, describedabove, were isolated by this technique.

Example 6 Enterokinase Cleavage of NMM-9ek

COS cells were transfected with plasmid pED.NMM9ek, which included thecDNA sequence of SEQ ID NO:17 as an insert. This construct contains anovel enterokinase cleavage site between the propeptide and maturepeptide of mocarhagin. After 48 hours, the transfected cells were washedin serum free medium, labelled with ³⁵S methionine for 6 hours, and theserum free conditioned mediuma was harvested. Purified bovineenterokinase (La Vallie et al., 1993, J. BIOL. Chem. 268:23311-23317)was added at various concentrations to 100 ul conditioned medium withlOmM Tris pH8 and InM CaCl₂, and incubate at 37C overnight. Soy TrypsinInhibitor resin was added to remove the enterokinase from the reactionmixture. The resin was pelleted by centrifugation and the supernatantwas then immunoprecipitated with rabbit polyclonal antibodies raisedagainst mocarhagin purified from cobra venom.

Following SDS-PAGE and autoradiography, a novel ˜50 kD band appeared inthe smple lane where 50 nanograms of purified bovine enterokinase hadbeen incubated with the conditioned medium (see FIG. 3). This band isconsistent with the expected molecular weight of the mature proteasewhen the propeptide (˜23 kD) is cleaved off.

1-95. (canceled)
 96. A method of treating an inflammatory diseasecomprising administering to a mammal a therapeutically effective amountof a composition comprising a mature mocarhagin protein comprising an Nterminal amino acid sequence chosen from: a) amino acids 192 to 221 ofSEQ ID NO: 6; b) amino acids 192 to 221 of SEQ ID NO: 8; c) amino acids192 to 221 of SEQ ID NO: 10; d) amino acids 192 to 221 of SEQ ID NO: 12;e) amino acids 192 to 221 of SEQ ID NO:
 14. 97. The method of claim 96,further comprising administering the composition with a pharmaceuticallyacceptable carrier.
 98. The method of claim 96, wherein the inflammatorydisease is chosen from arthritis, acute respiratory distress syndrome,asthma, emphysema, delayed type hypersensitivity reaction, systemiclupus erythmatosus, thermal injury, autoimmune thyroiditis, experimentalallergic encephalomyelitis, multiple sclerosis, diabetes, Reynaud'ssyndrome, neutrophilic dermatosis, inflammatory bowel disease, Grave'sdisease, glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 99. The method of claim 96, wherein themammal is a human.
 100. The method of claim 96, further comprisingadministering a cytokine, a lymphokine or hematopoietic factor.
 101. Themethod of claim 100, wherein the cytokine, lymphokine or hematopoieticfactor is chosen from M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,and erythropoietin.
 102. A method of treating an inflammatory diseasecomprising administering to a mammal a therapeutically effective amountof a composition comprising a mocarhagin protein comprising an aminoacid sequence chosen from: a) SEQ ID NO: 6; b) SEQ ID NO: 8; c) SEQ IDNO: 10; d) SEQ ID NO: 12; e) SEQ ID NO:
 14. 103. The method of claim102, further comprising administering the composition with apharmaceutically acceptable carrier.
 104. The method of claim 102,wherein the inflammatory disease is chosen from arthritis, acuterespiratory distress syndrome, asthma, emphysema, delayed typehypersensitivity reaction, systemic lupus erythmatosus, thermal injury,autoimmune thyroiditis, experimental allergic encephalomyelitis,multiple sclerosis, diabetes, Reynaud's syndrome, neutrophilicdermatosis, inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 105. The method of claim 102, wherein themammal is a human.
 106. The method of claim 102, further comprisingadministering a cytokine, a lymphokine or hematopoietic factor.
 107. Themethod of claim 106, wherein the cytokine, lymphokine or hematopoieticfactor is chosen from M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,and erythropoietin.
 108. A method of treating an inflammatory diseasecomprising administering to a mammal a therapeutically effective amountof a composition comprising a fragment of a mocarhagin protein, whereinthe fragment of the mocarhagin protein has mocarhagin activity, andcomprises a portion of any amino acid sequence chosen from: a) SEQ IDNO: 6; b) SEQ ID NO: 8; c) SEQ ID NO: 10; d) SEQ ID NO: 12; and e) SEQID NO:
 14. 109. The method of claim 108, wherein the fragment of themocarhagin protein is chosen from: a) amino acid 24 to amino acid 621 ofSEQ ID NO: 6; b) amino acid 192 to amino acid 621 of SEQ ID NO: 6; c)amino acid 24 to amino acid 439 of SEQ ID NO: 8; d) amino acid 192 toamino acid 439 of SEQ ID NO: 8; e) amino acid 24 to amino acid 613 ofSEQ ID NO: 10; f) amino acid 192 to amino acid 613 of SEQ ID NO: 10; g)amino acid 24 to amino acid 521 of SEQ ID NO: 12; h) amino acid 192 toamino acid 521 of SEQ ID NO: 12; i) amino acid 24 to amino acid 592 ofSEQ ID NO: 14; and j) amino acid 192 to amino acid 592 of SEQ ID NO: 14.110. The method of claim 108, further comprising administering thecomposition with a pharmaceutically acceptable carrier.
 111. The methodof claim 108, wherein the inflammatory disease is chosen from arthritis,acute respiratory distress syndrome, asthma, emphysema, delayed typehypersensitivity reaction, systemic lupus erythmatosus, thermal injury,autoimmune thyroiditis, experimental allergic encephalomyelitis,multiple sclerosis, diabetes, Reynaud's syndrome, neutrophilicdermatosis, inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 112. The method of claim 108, wherein themammal is a human.
 113. The method of claim 108, further comprisingadministering a cytokine, a lymphokine or hematopoietic factor.
 114. Themethod of claim 113, wherein the cytokine, lymphokine or hematopoieticfactor is chosen from M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,and erythropoietin.
 115. A method of inhibiting selectin-mediatedbinding comprising administering to a mammal a therapeutically effectiveamount of a composition comprising a mature mocarhagin proteincomprising an N terminal amino acid sequence chosen from: a) amino acids192 to 221 of SEQ ID NO: 6; b) amino acids 192 to 221 of SEQ ID NO: 8;c) amino acids 192 to 221 of SEQ ID NO: 10; d) amino acids 192 to 221 ofSEQ ID NO: 12; e) amino acids 192 to 221 of SEQ ID NO:
 14. 116. Themethod of claim 115, further comprising administering the compositionwith a pharmaceutically acceptable carrier.
 117. The method of claim115, wherein the mammal has a disease or condition chosen fromarthritis, acute respiratory distress syndrome, asthma, emphysema,delayed type hypersensitivity reaction, systemic lupus erythmatosus,thermal injury, autoimmune thyroiditis, experimental allergicencephalomyelitis, multiple sclerosis, diabetes, Reynaud's syndrome,neutrophilic dermatosis, inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 118. The method of claim 115, wherein themammal is a human.
 119. The method of claim 115, further comprisingadministering a cytokine, a lymphokine or hematopoietic factor.
 120. Themethod of claim 119, wherein the cytokine, lymphokine or hematopoieticfactor is chosen from M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,and erythropoietin.
 121. A method of inhibiting selectin-mediatedbinding comprising administering to a mammal a therapeutically effectiveamount of a composition comprising mocarhagin protein comprising anamino acid sequence chosen from: a) SEQ ID NO: 6; b) SEQ ID NO: 8; c)SEQ ID NO: 10; d) SEQ ID NO: 12; e) SEQ ID NO:
 14. 122. The method ofclaim 121, further comprising administering the composition with apharmaceutically acceptable carrier.
 123. The method of claim 121,wherein the mammal has a disease or condition chosen from arthritis,acute respiratory distress syndrome, asthma, emphysema, delayed typehypersensitivity reaction, systemic lupus erythmatosus, thermal injury,autoimmune thyroiditis, experimental allergic encephalomyelitis,multiple sclerosis, diabetes, Reynaud's syndrome, neutrophilicdermatosis, inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 124. The method of claim 121, wherein themammal is a human.
 125. The method of claim 121, further comprisingadministering a cytokine, a lymphokine or hematopoietic factor.
 126. Themethod of claim 125, wherein the cytokine, lymphokine or hematopoieticfactor is chosen from M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor,and erythropoietin.
 127. A method of inhibiting selectin-mediatedbinding comprising administering to a mammal a therapeutically effectiveamount of a composition comprising a fragment of a mocarhagin protein,wherein the fragment of the mocarhagin protein has mocarhagin activity,and comprises a portion of any amino acid sequence chosen from: a) SEQID NO: 6; b) SEQ ID NO: 8; c) SEQ ID NO: 10; d) SEQ ID NO: 12; and e)SEQ ID NO:
 14. 128. The method of claim 127, wherein the fragment of themocarhagin protein is chosen from: a) amino acid 24 to amino acid 621 ofSEQ ID NO: 6; b) amino acid 192 to amino acid 621 of SEQ ID NO: 6; c)amino acid 24 to amino acid 439 of SEQ ID NO: 8; d) amino acid 192 toamino acid 439 of SEQ ID NO: 8; e) amino acid 24 to amino acid 613 ofSEQ ID NO: 10; f) amino acid 192 to amino acid 613 of SEQ ID NO: 10; g)amino acid 24 to amino acid 521 of SEQ ID NO: 12; h) amino acid 192 toamino acid 521 of SEQ ID NO: 12; i) amino acid 24 to amino acid 592 ofSEQ ID NO: 14; and j) amino acid 192 to amino acid 592 of SEQ ID NO: 14.129. The method of claim 127, further comprising administering thecomposition with a pharmaceutically acceptable carrier.
 130. The methodof claim 127, wherein the mammal has a disease or condition chosen fromarthritis, acute respiratory distress syndrome, asthma, emphysema,delayed type hypersensitivity reaction, systemic lupus erythmatosus,thermal injury, autoimmune thyroiditis, experimental allergicencephalomyelitis, multiple sclerosis, diabetes, Reynaud's syndrome,neutrophilic dermatosis, inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's decease, necrotizingenterocolitis, granulocyte transfusion associated syndrome, andcytokine-induced toxicity.
 131. The method of claim 127, wherein themammal is a human.
 132. The method of claim 127, further comprising acytokine, a lymphokine or hematopoietic factor.
 133. The method of claim132, wherein the cytokine, lymphokine or hematopoietic factor is chosenfrom M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, stem cell factor, anderythropoietin.